The molybdenum storage protein from Azotobacter vinelandii has been purified to homogeneity. It has a molecular weight of 90,000 and contains 20 atoms of Mo per molecule. Mo in the storage protein is transferred readily to nitrogenase. The tungsten-containing analog of the iron-molybdenum cofactor (FeMo-co) can be activated in vitro by addition of molybdate to an extract. A new enzyme is required for this activity. By an extraction of FeMo-co with methyl ethyl ketone (MEK), we isolate a metal cluster that has lost 2 Fe atoms. This cluster of 6Fe:1Mo still exhibits the g equals 3.65 EPR signal and it also is capable of catalyzing acetylene reduction to ethylene. A flavoprotein, coded by nifF, specifically transfers electrons to nitrogenase (and therefore to FeMo-co) in Klebsiella pneumoniae. The flavoprotein has a molecular weight of 22,000 and contains FMN. Other electron transfer proteins previously have been reported to donate electrons to nitrogenase in vitro; however, our experiments indicate that these proteins have no specific function for nitrogen fixation in vivo.